The present invention relates to a system and method for isolating and purifying a biological sample and for testing the biological sample, and, more particularly, to a high-throughput, random access system and method for isolating and purifying nucleic acid contained in a biological sample and for testing the biological sample, such as by polymerase chain reaction testing.
Polymerase chain reaction (PCR) testing is a universally-accepted and widely-practiced laboratory method for replicating or amplifying the concentration of nucleic acid (NA), such as DNA, in a test tube. Replication/amplification takes place in an aqueous solution containing a concentration of DNA molecules. Pre-determined amounts of the polymerase enzyme, oligonucleotide primers, tri-phosphates of the four nucleic acids or substrates, activators, and stabilizers are then added to the aqueous solution, which is then subject to three thermal cycles, referred to as the denaturing cycle, the annealing cycle, and the elongation cycle.
During the first, denaturing cycle, the DNA double helix in aqueous solution is melted between about 90 and 95 degrees Centigrade (° C.) so that each strand of the double helix is separated from the other. During the second, annealing cycle, the denatured aqueous solution is cooled to a temperature between about 50 and about 65° C., causing the oligonucleotide primers to attach to complementary nucleotide sequences of each denatured DNA strand. Finally, during the elongation cycle, DNA double helixes are re-formed by elongation of the primers at a temperature between about 70 and about 72° C. More specifically, a thermostable polymerase, such as Taq-polymerase, bonds nucleotides to the primer templates attached to the complementary nucleotide sequences, which forms two new DNA double helixes where before there was just one. Accordingly, with every complete cycle, there is a doubling of the number of DNA molecules, so, the number of DNA molecules after n cycles is equal to 2n.
The duration of each of the three thermal cycles is very brief and typically measured in seconds. For example, the DNA molecules melt instantaneously at about 95° C. during the denaturing cycle. If the primers are available in sufficient concentration, primer hybridization during the annealing cycle only requires about one (1) or two (2) seconds. Finally, re-formation during the elongation cycle can occur at a bonding rate of about 80 per second. Hence, the elongation cycle needs only about two (2) seconds. Thus, theoretically, each PCR cycle requires about five (5) seconds to complete.
In practice, however, the duration of each thermal cycle depends on the rate of heat transfer, to heat or cool the aqueous solution at the pre-determined thermal cycle temperature. Variables that can affect the heating/cooling rates include, inter alia, the volume of the solution, the concentration of the aqueous solution, the thermal conductivity of the vessel holding the NA in aqueous solution, the thermal conductivity of the apparatus holding the vessel, and the method of applying and removing heat, e.g., by conduction or convection.
Conventionally, PCR testing is performed in “batches”. For example, typically, a thermal cycling device, such as a PCR plate, holds 96 vessels in a closely-spaced, 8×12 vessel-well pattern. Batch processing and large thermal cycling devices used for batch processing, however, have several shortcomings.
First, testing is not begun until each well in the thermal cycling device is filled with a vessel, which adds time. Second, because the pre-determined temperatures for the annealing and elongation thermal cycles vary depending on the nucleic acid being tested for, such as HIV, HCV, HDB, and so forth, testing is not begun until each well in the thermal cycling device is filled with a vessel containing an aqueous solution having a concentration of the same DNA molecule, which adds even more time. Third, because the vessels and thermal cycling device are introduced at once as a unit, the amount of time to bring the vessels and thermal cycling device to the pre-determined temperature associated with the thermal cycle will be greater than the amount of time to bring an individual vessel to the pre-determined temperature associated with the thermal cycle.
These shortcomings of batch processing can be addressed by a system and method that provide random access to each of the thermal cycles and that, moreover, provide individual temperature control over each vessel containing an aqueous solution.
U.S. Pat. No. 6,558,947 to Lund, et al. discloses a batch-type, thermal cycling device that enables one to control the temperature of each vessel well in the device independently of the temperature of adjacent vessel wells. This, however, only addresses half the problem because time is still spent filling up each of the 96 vessel wells before the thermal cycling device is batched.
Therefore, it would be desirable to provide a high-throughput system and testing method that provide random access to each of the thermal cycles and that provide individual temperature control over each vessel containing an aqueous solution.